Decellularized biologically-engineered tubular grafts

ABSTRACT

This disclosure describes decellularized, biologically-engineered tubular grafts and methods of making and using such decellularized, biologically-engineered tubular grafts.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims the benefit of priorityunder 35 U.S.C. §121 to, U.S. application Ser. No. 13/771,676 filed Feb.20, 2013, which claims the benefit of priority under 35 U.S.C. §119(e)to U.S. Application No. 61/691,394 filed Aug. 21, 2012.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HL083880,HL071538, and HL107572 awarded by National Institutes of Health Heart,Lung and Blood Institute (NHLBI). The government has certain rights inthe invention.

TECHNICAL FIELD

This disclosure generally relates to decellularized tubular grafts andmethods of making and using such decellularized tubular grafts.

BACKGROUND

Cardiovascular disease is the leading cause of mortality in the world:approximately one million surgical procedures are performed annually inthe US alone. Vascular grafts made from synthetic polyesters have shownsuccess in replacement of large diameter vessels such as the thoracicand abdominal aortas, aortic arch vessels, as well as the iliac andfemoral arteries. However, they have generally proven inadequate assmall-diameter (<6 mm) arterial grafts. This is primarily a result ofacute graft thrombogenicity, anastomotic intimal hyperplasia, aneurysmformation and infection. Autologous arteries and veins remain thestandard of care. However, a significant fraction of patients do nothave a suitable vessel that can be harvested as a replacement and donorsite morbidity occurs. Therefore, tissue engineering provides a viablealternative to create arterial grafts that can maintain vascularfunction comparable to native vessels.

SUMMARY

This disclosure describes decellularized tubular grafts and methods ofmaking and using such decellularized tubular grafts.

In one aspect, a decellularized, biologically-engineered tubular graftis provided. Such a biologically-engineered tubular graft includescell-produced extracellular matrix, wherein fibers within thebiologically-engineered tubular graft are aligned circumferentially.Such a decellularized, biologically-engineered tubular graft exhibitsgreater tensile stiffness in the circumferential direction than in thelongitudinal direction and exhibits a burst pressure that statisticallysignificantly exceeds a physiological pressure. In another aspect, abiologically-engineered tubular valve is provided that includes adecellularized, biologically-engineered tubular graft.

The cell-produced extracellular matrix can originate from a compositionthat includes matrix-producing cells, fibrinogen or a fibrinogen-likematerial, and thrombin. In some embodiments, the matrix-producing cellsare fibroblasts such as human dermal fibroblasts.

In some embodiments, the tubular graft exhibits a burst pressure of atleast 2500 mm Hg, at least 3000 mm Hg, at least 3500 mm Hg, or at least4000 mm Hg. In some embodiments, the tubular graft has an averagediameter of about 0.5 mm to about 6 mm, about 5 mm to about 12 mm, about10 mm to about 20 mm, or about 18 mm to about 24 mm. Representativetubular grafts include, without limitation, a vascular graft (e.g., anarterial graft or a venous graft), a urethra graft, a fallopian tubegraft, a Vas deferens graft, or a Eustachian tube graft.

In another aspect, a method of making the decellularized,biologically-engineered tubular graft described herein is provided. Sucha method typically includes combining fibrinogen or fibrinogen-likematerial, thrombin, and matrix-producing cells to produce a cell-seededfibrin gel; molding the cell-seeded fibrin gel into the shape of ahollow tube; manipulating, mechanically, the tube in the presence ofculture medium to produce a biologically-engineered tubular graft; anddecellularizing the biologically-engineered tubular graft. In someembodiments, such a method further can include anchoring one end of thedecellularized, biologically-engineered tubular graft at two positions,three positions, or four positions to shape the decellularized,biologically-engineered tubular graft into a bi-, tri- or quad-leafletvalve, respectively. In still another aspect, a biologically-engineeredvalve made by such a method is provided.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the methods and compositions of matter belong. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the methods and compositionsof matter, suitable methods and materials are described below. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing a representative method of using adecellularized, biologically-engineered tubular graft to make abiologically-engineered valve.

FIG. 2 is a schematic showing a representative method of using adecellularized, biologically-engineered tubular graft to make abiologically-engineered valve. The anchor points in FIG. 2 are createdby suturing one tube within another.

FIG. 3 is a schematic, including photographs, showing a representativemethod of using a decellularized biologically-engineered tubular graftto make a biologically-engineered valve. A frame in FIG. 3 provides theanchor points.

DETAILED DESCRIPTION

This disclosure describes a method of making the decellularized,biologically-engineered tubular graft and also describes thesubsequently produced decellularized, biologically-engineered tubulargraft. Initially, fibrinogen or fibrinogen-like material, thrombin, andmatrix-producing cells are combined, and the resulting cell-seeded gelcomposition is molded into the shape of a hollow tube. The fibrin-basedgel is remodeled into an extracellular matrix by the matrix-producingcells, which retains the tubular shape of the original mold.

Fibrinogen is a well known soluble glycoprotein naturally found inblood, and is converted to fibrin by the action of thrombin, a serineprotease, during blood coagulation. As used herein, fibrinogen-likematerial refers to proteins, natural or synthetic, that have similarcharacteristics to those of fibrinogen and, which, as described herein,can be converted into a fibrin-like material and, eventually, into anextracellular matrix material. One example of a fibrinogen-like materialis PEGylated-fibrinogen (see, for example, Suggs et al., 1998, J.Biomater. Sci. Polym. Ed., 9:653-66).

As used herein, “matrix-producing cells” are those cells that have thecapability of converting fibrinogen or a fibrinogen-like material, inthe presence of thrombin, into extracellular matrix. As describedherein, fibroblast cells are very efficient at producing extracellularmatrix material as are smooth muscle cells. Fibroblasts are well knownand can be human fibroblasts, primate fibroblasts, rodent fibroblasts,or any other type of mammalian fibroblasts. Fibroblasts can be obtainedfrom, for example, any number of different anatomical tissues (e.g.,dermis, lung, connective tissue, kidney, etc.), commercial sources(e.g., Millipore, Cell Applications, StemGent, etc.), and/or biologicaldepositories (e.g., American Type Culture Collection (ATCC)). The sameis true for smooth muscle cells. The matrix-producing cells can becombined with the fibrinogen or fibrinogen-like material, and thethrombin and then molded, or the matrix-producing cells can be seededinto a composition that includes fibrinogen or fibrinogen-like materialand thrombin and that has been molded into a tubular shape.

While matrix-producing cells at any passage beyond three passages (e.g.,four, five, six, seven, eight, nine, or ten passages) can be used incombination with the fibrinogen or fibrinogen-like material, it ispreferred that the matrix-producing cells that are used have been grownfor between three passages and seven passages. For example, fibroblastscan be grown for between three passages and six passages, for betweenfour passages and six passages, for between four passages and sevenpassages, or for between five passages and seven passages. In addition,the number of matrix-producing cells used initially in a tubular graftas described herein can be, for example, from about 10² cells to about10¹² cells (e.g., about 10³ cells to about 10¹⁰ cells, about 10⁴ cellsto about 10⁹ cells, 10⁵ cells to about 10⁸ cells, or about 10⁶ cells toabout 10⁷ cells).

Hydrogels are well known in the art and generally refer to hydrophilicpolymeric chains that can contain natural or synthetic polymers. One ormore hydrogels optionally can be included in the composition. Any numberof suitable hydrogels can be used, if desired, to support and mold thecell-seeded fibrin gel (e.g., comprising fibrinogen or fibrinogen-likematerial and the matrix-producing cells) as necessary. Representativehydrogels include, for example, agarose, methylcellulose, hyaluronan,and combinations thereof. If present, the amount of hydrogel used in acomposition described herein can range from about 10% to about 80%(e.g., about 20% to about 70%, about 30% to about 60%, about 40% toabout 50%, or about 50%). The presence of and the actual amount of oneor more hydrogels will depend upon the desired features of thecell-seeded fibrin gel or the subsequently producedbiologically-engineered tubular graft (e.g., softness or hardness,flexibility, absorbency).

The cell-seeded gel can be molded around, for example, a mandrel, inorder to obtain a hollow tube. Mandrels of different sizes can be useddepending upon the desired size of the hollow tube. For example, atubular graft as described herein can have a diameter of about 0.5 mm upto a diameter of about 24 mm (e.g., about 1 mm, about 2 mm, about 5 mm,about 10 mm, about 15 mm, about 20 mm, about 21 mm, about 22 mm, about23 mm, or about 24 mm in diameter; about 0.5 mm to about 6 mm, about 5mm to about 12 mm, about 10 mm to about 18 mm, about 15 mm to about 21mm, about 20 mm to about 24 mm, or about 18 mm to about 24 mm indiameter). In some embodiments, the diameter of the tubular graft can bean appropriate size for use as a vascular graft (e.g., an arterial orvenous graft, or arterio-venous fistula). For example and withoutlimitation, the diameter of the tubular graft can be an appropriate sizefor use as a graft for a urethra, a fallopian tube, a Vas deferens, or aEustachian tube. In addition, the larger diameter tubular grafts can beused to encircle or ensheath a structure such as, without limitation, aheart valve construct. The length of the tubular graft will be anylength that is appropriate or necessary to graft. For example, a tubulargraft as described herein can be from about 3.0 mm up to about 20 cm inlength (e.g., about 3.0 mm to about 10.0 mm, about 5.0 mm to about 20.0mm, about 25.0 mm to about 5.0 cm, about 50.0 mm to about 10 cm, about10 cm to about 15 cm, about 10 cm to about 20 cm, about 15 cm to about20 cm in length).

This combination of materials, over time, is converted by the cells intoan extracellular matrix (referred to herein as a “cell-producedextracellular matrix”), typically in a controlled laboratory setting.While the tubular graft is undergoing this transformation from thecell-seeded gel into the cell-produced extracellular matrix, the tubulargraft can be mechanically manipulated (or “conditioned”) in any numberof fashions. Mechanical manipulations include, for example, staticculture on the mandrel, which, for a non-adhesive mandrel, leads tocircumferential alignment as the cells compact the gel, causing theaxial length to shorten, and circumferential stretching or distensionwhile providing for axial shortening to maintain the circumferentialalignment. Generally, such stretching or distortion is cyclic orperiodic. See, for example, Syedain et al., 2011, Biomaterials,32:714-22. Simply by way of example, mechanical manipulations of thecell-seeded gel as it is remodeled into a cell-produced extracellularmatrix can be performed using flow-stretch or pulsed flow-stretchmethods in any number of bioreactors.

Such mechanical manipulation typically is done in the presence ofculture medium in order to maintain cell viability during the conversionprocess. The culture medium is the same medium usually used to culturecells such as fibroblasts, and includes components such as, withoutlimitation, serum, amino acids, salts (e.g., calcium chloride, potassiumchloride, magnesium sulfate, sodium chloride, and/or monosodiumphosphate), glucose, and vitamins (e.g., folic acid, nicotinamide,riboflavin, and/or B₁₂).

The biologically-engineered tubular graft then can be decellularized.Decellularization can take place using any number of different methods.See, for example, WO 2007/025233, WO 2010/120539, Ott et al. (2008, Nat.Med., 14:213-21), Baptista et al. (2009, Conf Proc. IEEE Eng. Med. Biol.Soc., 2009:6526-9) or Crapo et al. (2011, Biomaterials, 32:3233-43).

The resulting decellularized, biologically-engineered tubular graftexhibits a number of novel features. For example, the fibers are alignedcircumferentially around the tubular graft, which results in adecellularized, biologically-engineered tubular graft that exhibitsgreater tensile stiffness in the circumferential direction than in thelongitudinal direction. In addition, the decellularized,biologically-engineered tubular graft described herein exhibits a burstpressure that exceeds, and, in many cases, statistically significantlyexceeds, the pressure to which vasculature is normally or typicallyexposed (i.e., physiological pressure). For example, a decellularized,biologically-engineered tubular graft described herein can withstand apressure of at least 2000 mm Hg, at least 2500 mm Hg, at least 3000 mmHg, at least 3500 mm Hg, at least 4000 mm Hg, or greater than 4000 mmHg, without bursting. In other words, a decellularized,biologically-engineered tubular graft described herein can exhibit aburst pressure of at least 2000 mm Hg, at least 2500 mm Hg, at least3000 mm Hg, at least 3500 mm Hg, at least 4000 mm Hg, or greater than4000 mm Hg. Moreover, the cell-produced extracellular matrix isconducive to recellularization (i.e., cells, including endothelial cellspopulating the luminal surface and tissue cells penetrating andrepopulating the interior cavity) both in culture in the laboratory andendogenously following implantation or engraftment.

As indicated herein, a decellularized, biologically-engineered tubulargraft can be implanted or engrafted directly followingdecellularization. Alternatively, a decellularized,biologically-engineered tubular graft can be used to form atissue-engineered valve before being implanted or engrafted. Forexample, one end of a decellularized, biologically-engineered tubulargraft as described herein can be anchored at two points (for abi-leaflet valve), three points (for a tri-leaflet valve) or four points(for a quad-leaflet valve) using a suture, an adhesive, or othersuitable bonding method.

The schematic shown in FIG. 1 shows a structure in which three anchoringpoints are created. This leads to the formation of a structure, which,under fluid back-pressure, causes the tube to collapse and create avalvular action that is very similar to a tri-leaflet valve.Alternatively, FIG. 2 is a schematic showing an embodiment in which onetube is sutured to a tube placed over the first tube. As shown in FIG.2, suture lines are created that define three ‘U’ shapes around thecircumference of the tube. This leads to the formation of a structure asdescribed above that, under fluid back-pressure, causes the inner tubeto collapse and create a valvular action that is very similar to atri-leaflet valve.

In addition to the embodiment shown in FIG. 2, three points of anchoragecan be made using a material that is placed inside (e.g., a stent) oroutside (e.g., a frame) the tubular graft, which can be made of a rigidmaterial for a frame (e.g. titanium) or a flexible material for a stent(e.g. braided Nitinol wire mesh). FIG. 3 shows an embodiment in which atissue tube is placed on the outside of a rigid frame containing threestruts as anchoring points.

In addition to the anchoring methods described herein, which can be usedto make tissue-engineered valves, methods of modifying thebiologically-engineered tubular graft also are provided. For example, abiologically-engineered tubular graft can be formed with controlledrelease agents, or conjugated with biomolecules, or cross-linked, orrecellularized.

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1 Production and Implantation of Tubular Graft

Engineered grafts (4 mm ID, 2-3 cm long, 0.4 mm thickness) werefabricated from ovine dermal fibroblasts using the pulsed-flow-stretch(PFS) bioreactor and decellularized using sequential treatment with SDS,Triton-X, and DNase, which had little effect on graft properties. Thetotal graft culture time was 7 weeks. The burst pressure of thedecellularized grafts exceeded 4000 mm Hg and had the same compliance asthe ovine femoral artery. No cells were visible with histologicalstaining and DNA content was less than 10% of the untreated grafts. Thedecellularized grafts were implanted interpositionally in the femoral orcarotid artery of 4 sheep (n=6) for 8 weeks, in some cases thecontralateral position being used for a graft or sham control, where thenative artery segment being sutured back into place, and into 2 sheep(n=4) for 24 weeks. Anticoagulation therapy was used for the duration,but no immunosuppression was used.

Example 2 Physiology of Implanted Tubular Grafts

At both 8 and 24 weeks, all grafts were patent and showed no evidence ofdilatation or mineralization. Mid-graft lumen diameter was unchanged.Lumen diameter at the ends was 15% smaller for the grafts at 7 weeksbased on echocardiography, with a similar trend for the controls, butthis was not statistically significant at 24 weeks. A thin neointima wasevident near the ends of the grafts. Extensive recellularizationoccurred, with most cells expressing SMA. Deposition of organizedelastin was evident. Endothelialization was complete at the ends of thegrafts and partial mid-graft at 8 weeks and complete at 24 weeks.Complementary in vitro studies with a parallel plate flow chamber alsoindicate excellent shear resistance at physiological shear stresses ofpre-seeded blood outgrowth endothelial cells and mesenchymal stem cells.These studies indicate that the completely biological grafts possessingcircumferential alignment/tensile anisotropy, can be implanted into thearterial circulation without dilation or mineralization and minorintimal hyperplasia, and with favorable cell seeding, recellularization,and matrix remodeling.

Example 3 A Tissue-Engineered Tubular Valve

A tissue-engineered tubular graft having a lumen diameter of about 15-30mm was decellularized as described in Example 1. One end of the tube wasanchored at three points spaced approximately equally around thecircumference of the tube using a suture (as shown schematically in FIG.1).

FIG. 3 shows an embodiment in which a tissue tube is anchored around arigid frame. The tissue tube was sleeved over the outside of the frameand stitched along the three posts and around the bottom rim using aprolene suture. Under fluid back-pressure, the tissue tube collapsesbetween the three posts and creates a valvular action similar to atri-leaflet valve.

It is to be understood that, while the methods and compositions ofmatter have been described herein in conjunction with a number ofdifferent aspects, the foregoing description of the various aspects isintended to illustrate and not limit the scope of the methods andcompositions of matter. Other aspects, advantages, and modifications arewithin the scope of the following claims.

Disclosed are methods and compositions that can be used for, can be usedin conjunction with, can be used in preparation for, or are products ofthe disclosed methods and compositions. These and other materials aredisclosed herein, and it is understood that combinations, subsets,interactions, groups, etc. of these methods and compositions aredisclosed. That is, while specific reference to each various individualand collective combinations and permutations of these compositions andmethods may not be explicitly disclosed, each is specificallycontemplated and described herein. For example, if a particularcomposition of matter or a particular method is disclosed and discussedand a number of compositions or methods are discussed, each and everycombination and permutation of the compositions and the methods arespecifically contemplated unless specifically indicated to the contrary.Likewise, any subset or combination of these is also specificallycontemplated and disclosed.

1. (canceled)
 2. A method of making the decellularized,biologically-engineered tubular graft of claim 1, comprising: combiningfibrinogen or fibrinogen-like material, thrombin, and matrix-producingcells to produce a cell-seeded fibrin gel; molding the cell-seededfibrin gel into the shape of a hollow tube; manipulating, mechanically,the tube in the presence of culture medium to produce a cell-producedextracellular matrix; and decellularizing the biologically-engineeredtubular graft to produce a decellularized, biologically-engineeredtubular graft.
 3. The method of claim 2, wherein the matrix-producingcells are fibroblasts.
 4. The method of claim 2, wherein thematrix-producing cells are grown for between three passages and sevenpassages before being combined with fibrinogen or fibrinogen-likematerial and thrombin to produce the cell-seeded fibrin gel.
 5. Themethod of claim 2, wherein the number of matrix-producing cells that iscombined with fibrinogen or fibrinogen-like material and thrombin toproduce the cell-seeded fibrin gel is between about 10² matrix-producingcells and about 10¹² matrix-producing cells.
 6. The method of claim 2,wherein the manipulation step comprises at least one condition selectedfrom the group consisting of static culture and circumferentialstretching.
 7. The method of claim 2, wherein the manipulation step isapplied cyclically.
 8. The method of claim 2, wherein thedecellularized, biologically-engineered tubular graft exhibits fibersthat are aligned circumferentially around the tubular graft, whichresults in greater tensile stiffness of the graft in the circumferentialdirection than in the longitudinal direction
 9. The method of claim 2,wherein the tubular graft exhibits a burst pressure of at least 2500 mmHg.
 10. The method of claim 2, wherein the tubular graft exhibits aburst pressure of at least 3000 mm Hg.
 11. The method of claim 2,wherein the tubular graft exhibits a burst pressure of at least 3500 mmHg.
 12. The method of claim 2, wherein the tubular graft is a vasculargraft.
 13. The method of claim 12, wherein the tubular graft is anarterial graft.
 14. The method of claim 12, wherein the tubular graft isa venous graft.
 15. The method of claim 2, wherein the tubular graft isselected from the group consisting of a urethra graft, a fallopian tubegraft, a Vas deferens graft, or a Eustachian tube graft.
 16. The methodof claim 2, wherein the tubular graft has an average diameter of about0.5 mm to about 6 mm.
 17. The method of claim 2, wherein the tubulargraft has an average diameter of about 5 mm to about 12 mm.
 18. Themethod of claim 2, wherein the tubular graft has an average diameter ofabout 10 mm to about 20 mm.
 19. The method of claim 2, furthercomprising anchoring one end of the decellularized,biologically-engineered tubular graft at two or more positions to shapethe decellularized, biologically-engineered tubular graft into a leafletvalve.
 20. The method of claim 19, wherein the anchoring is at twopositions to shape the decellularized, biologically-engineered tubulargraft into a bi-leaflet valve.
 21. The method of claim 19, wherein theanchoring is at three positions to shape the decellularized,biologically-engineered tubular graft into a tri-leaflet valve.
 22. Themethod of claim 19, wherein the anchoring is at four positions to shapethe decellularized, biologically-engineered tubular graft into aquad-leaflet valve.
 23. A biologically-engineered valve made by themethod of claim 19.